1. Field of the Invention
This invention pertains to the identification of methods for inhibiting .alpha.-glucosidase and .alpha.-mannosidase, as well as the identification of optically pure enatiomers which inhibit these enzymes. Specifically, a family of compounds, which includes the two enantiomers of avarol, including the natural (+)-avarol and the non-natural (-)-avarol, synthetically prepared, and derivatives are demonstrated to be potent, selective inhibitors of these important enzymes.
2. Introduction
The toxicity of quinones is well-documented.sup.1 and explains the mutagenicity and carcinogenicity of many aromatic organic compounds existing as natural products, synthetic medicines, and environmental pollutants. Through metabolic processes these compounds are converted to quinonoid species responsible for their toxic effects. Quinones are widely used as bactericides, fungicides, and clinically useful chemotherapeutic agents possessing antileukemic and antitumor activity. Studies conducted with simple achiral quinones (e.g. p-benzoquinone, chloranil) have suggested that their toxic activity can be attributed not only to their ability to undergo redox cycling but also to their potential binding and alkylation of nucleic acids and essential thiol and amino groups in proteins.sup.1. The former process involves the production of the malign species superoxide radical, hydrogen peroxide, and hydroxyl radical which are believed to cause oxidative stress in cells by damage inflicted on DNA. The latter reflects the electrophilic nature of the quinone moiety. Given the facile conversion of hydroquinones to quinones under aerobic conditions,.sup.2 it stands to reason that chiral substituents on a hydroquinone nucleus might impart a degree of selectivity to the interaction between the respective quinone and asymmetric cellular components such as nucleic acids and highly organized proteins.
Glycosyl hydrolases.sup.3 (glycosidases) are an important class of enzymes that catalyze the hydrolysis of glycosidic bonds in polysaccharides and glycoproteins. The generally-accepted mechanism.sup.4 for this hydrolysis is where general acid-base catalysis by key residues in the protein serves to effect the transformation. The glycosidases can be grouped into two broad classes depending on whether the hydrolysis reaction they catalyze leads to overall retention or inversion at the anomeric center of the hydrolysis site. Both cases involve direct participation of a nucleophile and a proton donor positioned on opposite sides of the bond to be hydrolyzed. With inverting glycosidases, the difference involves a larger separation between these catalytic residues (.about.5.5 angstroms for retaining enzymes vs. .about.9.5 angstroms for inverting enzymes) to accommodate a water molecule..sup.5
Given the importance of polysaccharides and glycoproteins in cell-cell and host-pathogene recognition and their implication in the control of biological events,.sup.6,7,8 the relevance of the potentiation of their synthesis and/or function has grown tremendously. The ability to inhibit the biosynthetic pathways to carbohydrates and carbohydrate-protein conjugates is significant in the study of cellular and extra-cellular events and in the development of antiviral,.sup.6 antidiabetic,.sup.7 and antitumor.sup.8 chemotherapeutic strategies. At least two .alpha.-glucosidase inhibitors are currently in clinical trials for treatment of HIV-associated AIDS.
The quest for therapeutic strategies against the human immunodeficiency virus (HIV) and its ultimate manifestation, AIDS, represents a monumental effort in contemporary medicine and chemistry. To date, more than a half-million people in America alone have contracted the AIDS virus and the number continues to grow at an alarming rate. Several FDA-approved drugs are now being used clinically in various combinations or "cocktails" to ward off the virus and its detrimental consequences. All the currently approved drugs target one of two key retroviral enzymes, reverse transcriptase or protease, which are essential for replication and survival of the virus.
Another promising strategy indirectly targets the initial association and recognition event between the HIV virus and the fated host cell. The CD4 surface protein has been shown to be a specific cellular receptor for HIV. Klatzman et al., Nature 310:767 (1984) and Dalgleish et al., Nature 310:763 (1984). The CD4 antigen is bound by the envelope glycoprotein gp 120, a heavily glycosylated surface protein expressed by HIV, in the virus-host cell association event leading to cell membrane fusion and infectivity. The successful synthesis of a functional gp 120 can be hampered by inhibiting glycohydrolase enzymes (glycosidases) that act as the protein tailors of the cell. These enzymes are responsible for the selective trimming of carbohydrate moieties from the glycosylated protein as it is synthesized within the cell. The inhibition of certain glycosidases has been shown to have a profound effect on both the cell surface expression and function and topology of glycoproteins. Nichols et al., Mol. Cell. Biol. 5:3467 (1985). Thus, inhibitors of certain glycosidases, namely those which have an impact on the production of a competent gp120 glycoprotein, are potential candidates for the therapeutic treatment of HIV infection.
Since its isolation in 1974 from the marine sponge Dysidea avara by Minale et al..sup.10 avarol has been the subject of numerous biological studies seeking a better understanding of how this compound and its corresponding quinone exhibit their potent biological effects:
in vitro and in vivo inhibition of microtubule polymerization.sup.11 PA1 highly selective cytotoxicity against L5178Y mouse lymphoma cells in vitro and in vivo.sup.2a PA1 in vitro inhibition of replication.sup.12 of HTLV-III/LAV (the etiologic agent of AIDS)
Additionally, both avarol and avarone have been shown to be neither direct nor indirect mutagens.sup.13 n the Ames-microsomal assay.sup.14 and possess antimutagenic activity through the inhibition of benzo[a]pyrene monooxygenase..sup.13 Thus, an interesting scenario is presented in which avarol and avarone combine potent cytotoxicity and antiviral properties with antimutagenic activity.
Accordingly, it remains an object of those of skill in the art to obtain potent, preferably selective, inhibitors of glycosyl hydrolases. It is a further desire to elucidate the mechanism of action of avarol and avarone. ##STR1##